Inverse PCR-based detection reveal novel mobile genetic elements and their associated genes in the human oral metagenome

The human oral cavity is one of the hotspots harboring multiple mobile genetic elements (MGEs), which are segments of DNA that can move either within bacterial genomes or between bacterial cells that can facilitate the spreading of genetic materials, including antimicrobial resistance genes. It is, therefore, important to investigate genes associated with the MGEs as they have a high probability of dissemination within the bacterial population under selective pressure from human activities. As one-third of oral bacteria are not yet culturable in the laboratory condition, therefore, in this work, it is aimed to detect and identify the genetic contexts of MGEs in the oral cavity through an inverse PCR (IPCR)-based approach on the oral metagenomic. The human oral metagenome was extracted from saliva samples collected from healthy individuals in Tromsø, Norway. The extracted DNA was partially digested with the HindIII restriction enzyme and self-circularized by ligation. DNA primers targeting each MGE were designed to amplify outwards from the MGEs and used for the IPCR on the circularized DNA products. The IPCR amplicons were cloned into a pCR-XL-2-TOP vector, screened, and sequenced. Out of 40 IPCR amplicons, we confirmed and verified the genetic contexts of 11 samples amplified with primers targeting integron gene cassettes (GCs), IS431 composite transposons, and Tn916 conjugative transposons (tet(M) and xis-int). Novel integron GCs, MGEs, and variants of Tn916 conjugative transposons were identified, which is the first report using the IPCR technique to detect the genetic contexts of MGEs in the oral metagenomic DNA. Supplementary Information The online version contains supplementary material available at 10.1186/s12903-022-02209-y.


Introduction
The human oral cavity is the second most complex microbial ecosystem in the human body, inhabited by a comprehensive bacterial flora consisting of more than 700 bacterial species, of which 100-200 usually varies between individuals [60,62]. The oral cavity has dynamic physio-chemical conditions as it is a major gateway to the human body, so the oral bacteria have to adapt to these changes from food, drink, air, and human-related behaviors such as brushing, smoking, and kissing [25,28,49]. Because of the high clearance rate of planktonic bacteria caused by the continuous flow of saliva in the oral cavity, most bacteria live as a structured community inside a matrix of extracellular polysaccharides (EPS) of a biofilm called dental plaque [5,23]. Biofilm is a wellknown virulence factor because of its protective and sustaining nature towards the inhabiting microbes.

Open Access
*Correspondence: Mohammed.Al-Haroni@uit.no Tansirichaiya et al. BMC Oral Health (2022) 22:210 Biofilm was also shown to facilitate a process called horizontal gene transfer (HGT) that bacteria use to exchange their genetic materials, including antimicrobial resistance genes (ARGs) [24,30,45]. There are three main types of HGT, including transformation, conjugation, and transduction. For example, a conjugative transposon Tn5397, conferring tetracycline resistance, was shown to be transferred from a nonoral Bacillus subtilis donor to an oral Streptococcus sp. in a mixed-species biofilm [46]. The dynamic change in the oral cavity also provides a selective pressure that drives HGT in the oral community, which is important for their adaptability and evolution, including the spreading of ARGs among the oral commensal and possibly to other pathogens.
HGT has been shown to be facilitated by mobile genetic elements (MGEs), which are segments of DNA that inherit the ability to translocate from one bacterial replicon to another. It could facilitate the transfer either within a bacterial cell or between two different cells. Integrative conjugative elements, including conjugative plasmids and conjugative transposons, can facilitate intercellular transfer through conjugation. Even though the other MGEs, such as insertion sequences (ISs), composite transposons, unit transposons, and integrons, could not facilitate the transfer between cells by themselves. They can move within the bacterial cell, inserting themselves onto ICEs for an intercellular transfer. IS elements are the simplest MGE, containing only gene(s) for their transposition [51]. Other MGEs can carry additional or accessory genes, including ARGs, as part of their structure, such as DNA fragment between two IS elements of composite transposons and gene cassette (GC) array of integrons [38,47,54,57,58].
Genes associated with MGEs have a higher probability to be disseminated in the bacterial population when the MGEs move. Previously, the spread of ARGs against lastresort drugs was reported to be mediated by MGEs in several studies such as, mcr-1 colistin resistance gene that can be mediated by various plasmid types (IncI2, IncHI2, and IncX4) and transposons (Tn6330 and Tn6390) [32,35,53,63]. For the oral cavity, several resistance genes have also been shown to be associated with MGEs, such as tet(M) tetracycline resistance genes in Tn916-family conjugative transposons, ddl6 D-cycloserine resistance gene in an integron GC, qrg Cetyltrimethylammonium bromide (CTAB) resistance gene on an IS1216 composite transposon, and knt kanamycin resistance gene on IS257 composite transposon [6,41,44,56,59].
As one-third of the oral bacteria have not been cultured in the laboratory condition yet [11,61], investigating of MGEs and their associated genes through a cultureindependent method like metagenomics would be suitable as it would allow us to study these genes from an entire oral microbiome. Previously, genes associated with integrons and composite transposons in the oral metagenomic DNA have been studied through a PCRbased metagenomic approach [56,56,59,59], [57,58]. These studies amplified and identified the genetic context of both MGEs by designing DNA primers to amplify in the outward direction from common features flanking the DNA carried by the MGEs (insertion sequences (ISs) for composite transposons and attC recombination sites for integrons).
However, not all MGEs have two common features flanking their cargo DNA, so their genetic context cannot be investigated by conventional PCR. An inverse PCR (IPCR) is a technique that could overcome this limitation as it involves digestion of original template DNA, selfligation (circularization) of the digested products, and uses these self-circularized DNA molecules as templates for PCR (Fig. 1). Using IPCR on oral metagenomic DNA would therefore allow us to investigate and identify the genetic context upstream and downstream from a known single section of MGEs, which have a high probability to be disseminated in the oral microbiome. It was previously used to investigate the genetic context of resistance genes in sediment metagenome, which they found novel MGEs, and new putative ARGs [37].
In this study, we aimed to investigate the genetic context of MGEs in the human oral cavity through an IPCR-based metagenomic approach. The presence of MGEs in the Norwegian oral metagenome was confirmed by using both previously published primers and newly designed primers. Another set of primers amplifying outwards from the MGEs were then designed and used for the IPCR on the self-circularized oral metagenomic DNA template. Several novel integron GCs and variants of Tn916 were identified.

Study population, Saliva sample collection and extraction of oral metagenomic DNA
Saliva samples were collected from 50 healthy volunteers between 21 and 65, both male and female, who visited the University Dental Clinic at UiT the Arctic University of Norway. The inclusion criteria include not having been treated with antibiotics in the last 3 months and not having chronic diseases. Participants were asked to abstain from drinking, eating, and brushing their teeth at least one hour before the saliva collection. Ethical approval was obtained from the Regional Committees for Medical and Health Research Ethics (REK) prior to the sample collection (Project number 2018/1373/REK nord). All participants were properly informed and gave their written consent. A paraffin gum was used to stimulate saliva secretion, and approximately 2 mL of saliva was collected from each participant using Norgen's Saliva DNA Collection, Preservation and Isolation Kit (Norgen Biotek Corp, Ontario, Canada). All the saliva samples were anonymized and stored at room temperature according to the manufacturer's instructions.

Extraction of the oral metagenomic DNA
750 µL of saliva from each sample was transferred to a 2-mL microcentrifuge tube and mixed with 750 µl of phosphate-buffered saline (PBS). Each tube was then centrifuged for 10 min at 15,700 ×g to pellet the cells. The supernatant was discarded and resuspended the pellet in 125 μl PBS buffer and 25 μl MetaPolyzyme (Sigma-Aldrich, Norway). After incubating the tubes at 35 °C for 4 h, oral metagenomic DNA was extracted from each saliva sample by QIAcube (Qiagen, Norway) and QIAamp ® DNA Mini QIAcube Kit. A pooled metagenomic DNA sample was prepared by aliquot and mixing 10 µl of each extracted oral metagenomic DNA in a new Eppendorf tube and kept at − 20 °C.

Confirmation of MGEs in oral metagenomic DNA
The presence of each MGE in oral metagenomic DNA was first determined by primers listed in Additional file 1: Table S1. Primers targeting plasmids, Tn916-family conjugative transposons, and integrons were selected from previously published studies that had been used to successfully amplify the MGEs based on their conserved genes/sequences. For composite transposons, a list of composite transposons associated with ARGs was Fig. 1 Schematic representation of IPCR-based approach to determine the genetic context of MGEs in the human oral metagenome. Saliva samples were collected from healthy individuals, and oral metagenomic DNA was subsequently extracted from each sample. The extracted metagenome was partially digested with HindIII restriction enzyme and self-ligated as circular DNA molecules to use as templates for IPCR. The unknown genetic context (blue open arrows) of each MGE (green arrows) can then be detected by designing DNA primers (red arrows) to amplify outwards from the MGE. The IPCR amplicons are cloned into the vector, sequenced, and analyzed by bioinformatics selected from the Transposon Registry [57,58]. Then, primers were designed to target the conserved regions of their IS elements, identified by aligning available gene sequences from the GenBank nucleotide database and ISFinder [52], using Clustal Omega and Primer3 webbased software (https:// bioin fo. ut. ee/ prime r3-0. 4.0/). PCR reactions of each primer pair were set up, containing 1 µl of the pooled oral metagenomic DNA, 2 µl of each primer (10 µM), 15 µl of 2 × Biomix red (Bioline, Norway), and 10 µl molecular grade water. The PCR cycle was programmed as follows: (i) initial denaturation at 95 °C for 5 min, (ii.) denaturation at 95 °C for 1 min, (iii.) annealing at 50-65 °C depending on the primers for 30 s, (iv.) elongation at 72 °C for 1 min 30 s, repeated step (ii.)-(iv.) for 35 cycles, and finished with a final elongation at 72 °C for 5 min. Amplicons from each PCR were cleaned using QIAquick PCR Purification Kit (Qiagen, Norway), following the protocol from the manufacturer. The presence of MGEs was confirmed by checking for the expected band of each MGE on agarose gels and by Sanger sequencing of the amplicons at the Genewiz, Germany.

Digestion and circularization of the oral metagenomic DNA
The pooled oral metagenomic DNA was partially digested with HindIII restriction enzyme at 37 °C for 2, 3, and 4 min. The digested products were run on an agarose gel, and DNA products larger than 1000 bp were cut out and extracted from the gel by using QIAgen gel extraction kit (QIAgen, Norway), following the instructions from the manufacturer. After the gel extraction, the DNA fragments were subjected to selfligation and formed as circular forms by setting up ligation reaction as follows: 1 µl T4 DNA ligase (0.05 U/ μL), 10 µl 10X T4 DNA ligase buffer, 50-100 ng of the digested DNA product and topped up with molecular grade water to a final volume of 100 µl. The self-ligation reaction was incubated at 16 °C overnight and purified by using QIAquick PCR Purification Kit, following the protocol from the manufacturer.

Amplification and cloning of the genetic context of the MGEs in the oral metagenomic DNA
The genetic context of the detected MGEs in the human oral metagenome was determined through the uses of IPCR primers (listed in Additional file 1: Table S1), amplifying outwards from the MGE into the flanking areas to use for the IPCR, which were designed by reverse complementing the MGE confirmation primers. The IPCR reactions consisted of 25 µl Platinum SuperFi Green PCR Master Mix (ThermoFisher, Norway), 2.5 µl of each inverse-PCR primers (10 µM), 5-50 ng of the self-circularized metagenomic DNA, and top up with molecular grade water to a final volume of 50 µl. The PCR cycle was programmed as follows: (i.) initial denaturation at 98 °C for 5 min, (ii.) denaturation at 98 °C for 1 min, (iii.) annealing at 50-65 °C depending on the primers for 30 s, (iv.) elongation at 72 °C for 6 min, repeated step (ii.)-(iv.) for 35 cycles, and finished with a final elongation at 72 °C for 5 min.
The amplicons were cloned into pCR ™ -XL-2-TOPO ™ vectors through TOPO cloning using the TOPO ™ XL-2 Complete PCR Cloning Kit (ThermoFisher, Norway), which is suitable for the cloning of long PCR products (up to 13 kbp), by following the protocol from the manufacturer. The ligated product was then transformed into One Shot ™ OmniMAX ™ 2 T1R chemically competent Escherichia coli cells using a standard heat shock protocol. The transformants were grown on LB agar supplemented with ampicillin (100 µg/ml) and incubated overnight at 37 °C.

Plasmid isolation and sequencing of the extracted plasmids
The transformants of each MGE were screened by performing colony PCR with T3 and T7 primers and checked the colony PCR products on agarose gels. Colonies with different insert sizes and larger than 1000 bp were selected and subcultured into 5 ml of LB broth supplemented with ampicillin (100 µg/ml), then incubated overnight in a 37 °C shaker (200 rpm). The extracted plasmids were isolated from the overnight culture by using QIAprep Spin Miniprep Kit (QIAgen, Norway). All plasmids were sequenced by Sanger sequencing service at the Genewiz, Germany, using M13 Forward and M13 Reverse as initial primers for sequencing. Additional primers were designed and used for additional sequencing for the samples with longer inserts by using Primer3 (https:// bioin fo. ut. ee/ prime r3-0. 4.0/).

Bioinformatics analysis
The sequencing data was visualized and assembled with CAP contig function by using BioEdit software version 7.2.0 (http:// www. mbio. ncsu. edu/ bioed it/ bioed it. html) [22]. The contamination of vector sequences was checked and removed by using the VecScreen analysis tool (https:// www. ncbi. nlm. nih. gov/ tools/ vecsc reen/). The presence of both forward and reverse primers, and the conserved sequences of each MGE were then identified by searching and comparing the sequences. For the integron gene cassette samples, additional criteria were applied, such as the presence of attC core sites, as described by previous studies [56][57][58][59]. The sequences were compared to the nucleotide and protein databases in GenBank with BlastN and BlastX, respectively [1]. All sequences detected in this study were deposited to the GenBank under accession numbers from OL695865 to OL695875.

Confirmation of MGEs in oral metagenomic DNA
A collection of MGEs, known to be associated with ARGs, was selected to be investigated in this study, including 4 Inc-type plasmids (IncP-1α, IncP-1β, IncP-9, and IncQ), Tn21-related transposons, Tn916-family conjugative transposons, integrons, and 17 composite transposons. Prior to the investigation of the genetic context of MGEs by IPCR, the presence of each MGEs in oral metagenomic DNA was determined through PCR using previously published primers, except for composite transposons, which were detected by using new primers designed to target the IS elements of each composite transposons (Additional file 1: Table S1). Sequencing the amplicons of the expected size confirmed the presence of 3 plasmid types, integrons, Tn21-related transposons, Tn916-family conjugative transposons, and 8 out of 17 IS elements in the oral metagenomic DNA (Table 1).

Identification and characterization of genes associated with MGEs via IPCR
Another set of primers was designed to amplify outward from the common features of the confirmed MGEs and used in the IPCR on the self-circularized oral metagenomic DNA. The amplicons from IPCR could be either the DNA region flanked by two repeats of MGEs (IS elements of composite transposons and attC recombination sites of integrons) or the upstream and downstream genetic contexts of a single conserved gene on MGEs.
Screening and bioinformatic analysis confirmed that 11 out of 40 IPCR DNA fragments as true amplicons, not PCR artifacts, with a size between 1.7 and 5 kb. The details and predicted genes on each IPCR amplicon are shown in Table 2. These samples could be divided into two groups. The first group was amplified from two DNA repeats, including 5 integron GC samples and 1 IS431 composite transposon (Fig. 2). All integron gene cassettes were predicted to derive from Treponema species, and most of them, except Flip-MARS-9, contained two GCs. Several proteins were predicted to be encoded by these integron GC samples, such as toxin-antitoxin proteins (Flip-MARS-4 and Flip-MARS-5), endonuclease enzymes (Flip-MARS-3 and Flip-MARS-4), carbon-nitrogen hydrolase (Flip-MARS-9), vicinal oxygen chelate (VOC) family protein, and competence protein TfoX (Flip-MARS-11). Also, IS4 family transposase was identified in the Flip-MARS-11 sample, which was similar to the transposase on ISPca1 with 36% identities based on the BlastX analysis on ISFinder. For the IS431-5 composite transposon sample, it was predicted to contain two hypothetical proteins.
The second group of samples was those amplified from a single conserved gene of MGEs on self-circularized DNA templates. It included the samples amplified by DNA primers targeting Tn916-family conjugative transposons: 4 tet(M) samples and 1 xis-int sample. (Fig. 3). HindIII restriction site, which was used for the self-circularization, was identified at the nucleotide position 230 of MFS transporter gene in xis-int-9 sample and the nucleotide position 63 of tet(M) gene of all tet(M) samples. tet(M)-6, tet(M)-9, and xis-int-9 were similar to integrative and conjugative elements ICESpnIC1, ICE6BST90 and Tn6822, respectively, which contained Tn916-related structures. For Tet(M)-2, it contained part of IS21-like helper ATPase, which was similar to the helper protein of ISCbe3 with 67% identities based on the BlastX analysis on ISFinder, while tet(M)-6 were shown to contain erythromycin resistance gene erm(B) next to a Tn3-family transposase and resolvase.

Discussion
Mobile genetic elements play a crucial role in the antimicrobial resistance crisis as they can facilitate the movement of ARGs in the bacterial population. The surveillance of ARGs and other genes associated with MGEs is therefore important as they are highly likely to be disseminated, especially with selective pressure from all uses of antimicrobials. As the oral cavity is the gateway of the human body connecting to other organs like the gastrointestinal tract, MGEs could promote not only the     movement of these genes from oral microflora to clinical pathogens but also have the potential to transfer their genes to other microbiomes as well. In our study, it is the first report that showed the uses of the IPCR-based approach to investigate the genetic contexts of MGEs in the oral metagenome, where several variants of Tn916 conjugative transposons and novel integron genes cassettes were identified. The uses of IPCR technique with metagenomic DNA has only been used previously to study the genetic context of resistance genes in the sediment metagenome, which was shown to be a cost-efficient method with higher sensitivity compared to the metagenomic sequencing approach [37]. With metagenomic sequencing, it requires the metagenome to have a deep enough sequencing depth to perform assembly properly, especially for the MGEs with low abundance, which increases the sequencing cost [3,4]. Also, the structures of MGEs, which contain both conserved and variable regions, and multiple insertions of the same MGEs in bacterial genomes or bacterial cells in an environment increase the challenges for the assembly algorithms. IPCR could bypass these issues as the target DNA are enriched by the amplification of the low abundance MGEs, and the cloning of IPCR amplicons into vector allows us to use primer walking for sequencing, which are cheaper and the sequencing data can be analyzed through simple bioinformatic tools.
The detected integron GC and composite transposon samples were amplified from two DNA repeats of IS elements and attC, respectively, which could be amplified either from undigested linear or selfcircularized oral metagenome. The amplification of genes between attC has been used commonly to investigate integron GCs in various environmental samples such as wastewater, soil, marine, and saliva [2,14,21,56,59]. All integron samples were related to Treponema species, corresponding to the design of MARS2 and MARS5 primers that was based on the attC sequence of the reverse integron of T. denticola ATCC 35,405 [9]. However, all samples detected by this primer pair in the previous studies contained single GC [56][57][58][59], but we identified 4 integron samples containing 2 GCs (including interspersed attC sites) in our study, which is the first time that double cassettes were recovered from the oral metagenome by PCR approach. It could occur from the additional gel extraction step to include only IPCR amplicons larger than 1 kb prior to the cloning step, which was not performed in the previous studies, in which the integron amplicons size from previous studies was 425-1263 bp, while the size of integron samples in this study was 1704-2890 bp.
Several PCR-based studies on integron in other environmental samples also recovered amplicons with multiple GCs previously marine sediment and deep-sea hydrothermal vent samples, where they found 44% and 32.5% of the samples to contain multiple GCs [13,14]. This is possible as integrons can carry from a dozen of GCs in mobile integrons to hundreds of GCs in sedentary chromosomal integrons where each GC is separated by attC recombination sites (59-120 bp) with similar sequences to each other [17]. Therefore, multiple GCs can be recovered if DNA primers bind to attC that are not adjacent but bind to the attC located further instead.
Analyzing the detected integron GCs from the oral metagenome predicted several interesting proteins encoding by these GCs. Toxin-antitoxin proteins were detected in Flip-MARS-4 and Flip-MARS-5 GCs, same as in the previous studies using the MARS2-MARS5 primers [56][57][58][59]. The presence of toxin-antitoxin containing GCs is common for sedentary chromosomal integrons like T. denticola integrons as their function is to prevent random deletion of GCs and ensure the stability of the large GC arrays [19,33,48,55]. Endonucleases were also predicted to be encoded by Flip-MARS-3 and Flip-MARS-4 GCs, which were also found in another study that performed an in silico analysis on metagenomic datasets of the Human Microbiome Project to identify integrons associated with Treponema spp [66]. The function of endonuclease enzymes in integrons has been suggested to involve in the cassette neoformation process [31]. Another gene, encoding VOC family protein, was also identified from Flip-MARS-11. Proteins in this family are metalloenzyme catalyzing diverse reactions through the formation of the partially closed beta-sheet barrel, which is formed by two βαβββ units, wrapping around the metal ions [20]. The important members of VOC family include glyoxalases I, fosfomycin resistance proteins and bleomycin resistance proteins [20,27,50].
Tn916-family conjugative transposons are one of the largest families of ICEs. They can be found either as a linear form in a bacterial genome or as a circular intermediate in which their backbones contain conjugative transfer, transcriptional regulation, and recombination modules. Many of the Tn916 members also carry tet(M) as part of the accessory module. Tn916, the first and smallest member of this family, was isolated from Enterococcus faecalis DS16 [15], which later has been found in over 30 different bacterial genera [6,43]. For the human oral cavity, Tn916-like elements have been found predominantly in oral streptococci, Veillonella spp., and Neisseria spp [29,34,42]. In our study, we used primers targeting tet(M) and xis-int genes on Tn916, which we identified 3 Tn916-like elements that were likely to derive from Streptococcus pneumoniae based on BlastN results.
The genes associated with the detected Tn916 variants were including erm(B) erythromycin resistance gene, Tn3 family transposase and resolvase genes (tnpA and tnpR), and MFS transporter gene. The presence of erm(B) has been found in several members of the Tn916 family, such as Tn3872, Tn1545, and Tn6003 [8,36]. However, the structure that tnpA and tnpR genes located downstream from erm(B) in tet(M)-6 sample was similar to a Tn917 unit transposon structure, and the only transposon in the transposon registry that Tn917 located next to tet(M) was Tn3872 [36]. The presence of Tn917 containing erm(B) on Tn916-family conjugative transposon represents the phenomenon where ARGs could be moved onto ICEs with the help of other non-conjugative elements, allowing these ARGs to be subsequently moved to other bacterial cells by the activity of ICEs. For the MFS transporter in the xis-int-9 sample, it is an efflux pump, which can be found in all phyla, from bacteria to mammals [39]. Their main function involves the uptake of sugars, but some are also involved in multidrug resistance (MDR), virulence, and biofilm formation in bacteria [40]. Examples of MFS transporter conferring MDR are LmrP protein in Lactococcus lactis conferring lincosamides, macrolides and streptogramins resistance, and MdfA protein in E. coli conferring chloramphenicol, erythromycin, ciprofloxacin and rifampicin resistance [10,65].
MGEs were also identified in the detected MGEs, including IS4-family transposase in the Flip-MARS-11 sample and Tn3-family tnpA-tnpR in the tet(M)-6 sample. As the core structures of integrons do not have genes to catalyze their interchromosomal mobility, they have to rely on transposase or recombinase from other sources [16]. The IS4-family transposase detected in Flip-MARS-11 could be kept in an integron cassette array in the oral cavity for such a purpose as well. It is also the first time that IS element was found in a cassette array in the oral metagenome. The presence of transposase genes in cassette array was previously reported in Xanthomonas integrons [18]. For the tet(M)-2 sample, even though it was not amplified from Tn916-like elements, it contained the IS21-family helper protein next to the tet(M) gene. Therefore, there was a high probability for the tet(M) in this sample to be disseminated with the help of its associated IS21-family element, similar to the previous report that showed that IS21-558 involved in the movement of cfr multidrug resistance gene [26].
Our study showed that the human oral microbiome is also one of the hot spots that contained a diverse pool of MGEs, which is consistent with other previous studies, where each MGE can be associated with various genes. For example, previous studies reported several different structures of IS1216 composite transposons in the oral microbiome, containing qrg antiseptic resistance gene and universal stress protein A (uspA) gene [6,56,59]. Multiple Tn916-Tn1545 family conjugative transposons were also identified from the oral bacteria, especially Streptococcus spp. such as Tn6002 from Streptococcus cristatus, Tn6815 from Streptococcus mitis, and Tn6816 from Streptococcus constellatus [7,34,64], where their transferability between oral streptococci were also confirmed. As all uses of antimicrobials provide selective pressure for bacteria to develop and increase the transfer of these MGEs, the oral microbiome, as a major gateway to the whole body, have a high potential to act as a reservoir for the spread of antimicrobial resistance to other microbiomes through a myriad of MGEs [12]. Therefore, the oral microbiome is an important part that needs to be understood on a functional level for us to fully understand the antibiotic resistance crisis.

Conclusion
To summarize, we have determined that genes associated with MGEs in the oral metagenome can be investigated through an IPCR-based approach, which requires less cost and simpler bioinformatic analysis compared to the metagenomic sequencing approach. As a result, several novel integron GCs, novel MGEs, and variants of Tn916 were recovered from the oral metagenome.